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Total plasma protein N -glycosylation (TPNG) changes are a hallmark of many diseases. Here, we analyzed the TPNG of 169 COVID-19 patients and 12 healthy controls, using mass spectrometry, resulting in the relative quantification of 85 N -glycans. We found a COVID-19 N -glycomic signature, with 59 glycans differing between patients and controls, many of them additionally differentiating between severe and mild COVID-19. Tri- and tetra-antennary N -glycans were increased in patients, showing additionally elevated levels of antennary α2,6-sialylation. Conversely, bisection of di-antennary, core-fucosylated, nonsialylated glycans was low in COVID-19, particularly in severe cases, potentially driven by the previously observed low levels of bisection on antibodies of severely diseased COVID-19 patients. These glycomic changes point toward systemic changes in the blood glycoproteome, particularly involvement of acute-phase proteins, immunoglobulins and the complement cascade. Further research is needed to dissect glycosylation changes in a protein- and site-specific way to obtain specific functional leads., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Background: Changes in plasma protein glycosylation are known to functionally affect proteins and to associate with liver diseases, including cirrhosis and hepatocellular carcinoma. Autoimmune hepatitis (AIH) is a liver disease characterized by liver inflammation and raised serum levels of IgG, and is difficult to distinguish from other liver diseases. The aim of this study was to examine plasma and IgG-specific N-glycosylation in AIH and compare it with healthy controls and other liver diseases., Methods: In this cross-sectional cohort study, total plasma N-glycosylation and IgG Fc glycosylation analysis was performed by mass spectrometry for 66 AIH patients, 60 age- and sex-matched healthy controls, 31 primary biliary cholangitis patients, 10 primary sclerosing cholangitis patients, 30 non-alcoholic fatty liver disease patients and 74 patients with viral or alcoholic hepatitis. A total of 121 glycans were quantified per individual. Associations between glycosylation traits and AIH were investigated as compared to healthy controls and other liver diseases., Results: Glycan traits bisection (OR: 3.78 [1.88-9.35], p-value: 5.88 × 10 - 3 ), tetraantennary sialylation per galactose (A4GS) (OR: 2.88 [1.75-5.16], p-value: 1.63 × 10 - 3 ), IgG1 galactosylation (OR: 0.35 [0.2-0.58], p-value: 3.47 × 10 - 5 ) and hybrid type glycans (OR: 2.73 [1.67-4.89], p-value: 2.31 × 10 - 3 ) were found as discriminators between AIH and healthy controls. High A4GS differentiated AIH from other liver diseases, while bisection associated with cirrhosis severity., Conclusions: Compared to other liver diseases, AIH shows distinctively high A4GS levels in plasma, with potential implications on glycoprotein function and clearance. Plasma-derived glycosylation has potential to be used as a diagnostic marker for AIH in the future. This may alleviate the need for a liver biopsy at diagnosis. Glycosidic changes should be investigated further in longitudinal studies and may be used for diagnostic and monitoring purposes in the future., (© 2024. The Author(s).)

The asialoglycoprotein receptor (ASGPR) and the mannose receptor C-type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N-Acetylgalactosamine are recognized by ASGPR, while terminal mannose, fucose, and N-Acetylglucosamine are recognized by MRC1. The effects of ASGPR and MRC1 deficiency on the N-glycosylation of individual circulating proteins have been studied. However, the impact on the homeostasis of the major plasma glycoproteins is debated and their glycosylation has not been mapped with high molecular resolution in this context. Therefore, we evaluated the total plasma N-glycome and plasma proteome of ASGR1 and MRC1 deficient mice. ASGPR deficiency resulted in an increase in O-acetylation of sialic acids accompanied by higher levels of apolipoprotein D, haptoglobin, and vitronectin. MRC1 deficiency decreased fucosylation without affecting the abundance of the major circulating glycoproteins. Our findings confirm that concentrations and N-glycosylation of the major plasma proteins are tightly controlled and further suggest that glycan-binding receptors have redundancy, allowing compensation for the loss of one major clearance receptor., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Visceral leishmaniasis (VL) is a clinical form of leishmaniasis with high mortality rates when not treated. Diagnosis suffers from invasive techniques and sub-optimal sensitivities. The current (affordable) treatment with pentavalent antimony as advised by the WHO is possibly harmful to the patient. There is need for an improved diagnosis to prevent possibly unnecessary treatment. N -glycan analysis may aid in diagnosis. We evaluated the N -glycan profiles from active VL, asymptomatic infections (ASYMP) and controls from non-endemic (NC) and endemic (EC) areas. Active VL has a distinct N -glycome profile that associates with disease severity. Our study suggests that the observed glycan signatures could be a valuable additive to diagnosis and assist in identifying possible markers of disease and understanding the pathogenesis of VL. Further studies are warranted to assess a possible future role of blood glycome analysis in active VL diagnosis and should aim at disease specificity., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Breast cancer is the most prevalent cancer in women. Early detection of this disease improves survival and therefore population screenings, based on mammography, are performed. However, the sensitivity of this screening modality is not optimal and new screening methods, such as blood tests, are being explored. Most of the analyses that aim for early detection focus on proteins in the bloodstream. In this study, the biomarker potential of total serum N-glycosylation analysis was explored with regard to detection of breast cancer. In an age-matched case-control setup serum protein N-glycan profiles from 145 breast cancer patients were compared to those from 171 healthy individuals. N-glycans were enzymatically released, chemically derivatized to preserve linkage-specificity of sialic acids and characterized by high resolution mass spectrometry. Logistic regression analysis was used to evaluate associations of specific N-glycan structures as well as N-glycosylation traits with breast cancer. In a case-control comparison three associations were found, namely a lower level of a two triantennary glycans and a higher level of one tetraantennary glycan in cancer patients. Of note, various other N-glycomic signatures that had previously been reported were not replicated in the current cohort. It was further evaluated whether the lack of replication of breast cancer N-glycomic signatures could be partly explained by the heterogenous character of the disease since the studies performed so far were based on cohorts that included diverging subtypes in different numbers. It was found that serum N-glycan profiles differed for the various cancer subtypes that were analyzed in this study.

Apolipoprotein-CIII (apo-CIII) is a glycoprotein involved in lipid metabolism and its levels are associated with cardiovascular disease risk. Apo-CIII sialylation is associated with improved plasma triglyceride levels and its glycosylation may have an effect on the clearance of triglyceride-rich lipoproteins by directing these particles to different metabolic pathways. Large-scale sample cohort studies are required to fully elucidate the role of apo-CIII glycosylation in lipid metabolism and associated cardiovascular disease. In this study, we revisited a high-throughput workflow for the analysis of intact apo-CIII by ultrahigh-resolution MALDI FT-ICR MS. The workflow includes a chemical oxidation step to reduce methionine oxidation heterogeneity and spectrum complexity. Sinapinic acid matrix was used to minimize the loss of sialic acids upon MALDI. MassyTools software was used to standardize and automate MS data processing and quality control. This method was applied on 771 plasma samples from individuals without diabetes allowing for an evaluation of the expression levels of apo-CIII glycoforms against a panel of lipid biomarkers demonstrating the validity of the method. Our study supports the hypothesis that triglyceride clearance may be regulated, or at least strongly influenced by apo-CIII sialylation. Interestingly, the association of apo-CIII glycoforms with triglyceride levels was found to be largely independent of body mass index. Due to its precision and throughput, the new workflow will allow studying the role of apo-CIII in the regulation of lipid metabolism in various disease settings., Competing Interests: D.D. is employed by Ludger Ltd. and V.D. is employed by Janssen Vaccines & Prevention. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Demus, Naber, Dotz, Jansen, Bladergroen, Nouta, Sijbrands, Van Hoek, Nicolardi and Wuhrer.)

Background &aims: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer type with loco-regional spread that makes the tumor surgically unresectable. Novel diagnostic tools are needed to improve detection of PDAC and increase patient survival. In this study we explore serum protein N-glycan profiles from PDAC patients with regard to their applicability to serve as a disease biomarker panel., Methods: Total serum N-glycome analysis was applied to a discovery set (86 PDAC cases/84 controls) followed by independent validation (26 cases/26 controls) using in-house collected serum specimens. Protein N-glycan profiles were obtained using ultrahigh resolution mass spectrometry and included linkage-specific sialic acid information. N-glycans were relatively quantified and case-control classification performance was evaluated based on glycosylation traits such as branching, fucosylation, and sialylation., Results: In PDAC patients a higher level of branching (OR 6.19, P-value 9.21 × 10 -11 ) and (antenna)fucosylation (OR 13.27, P-value 2.31 × 10 -9 ) of N-glycans was found. Furthermore, the ratio of α2,6- vs α2,3-linked sialylation was higher in patients compared to healthy controls. A classification model built with three glycosylation traits was used for discovery (AUC 0.88) and independent validation (AUC 0.81), with sensitivity and specificity values of 0.85 and 0.71 for the discovery set and 0.75 and 0.72 for the validation set., Conclusion: Serum N-glycome analysis revealed glycosylation differences that allow classification of PDAC patients from healthy controls. It was demonstrated that glycosylation traits rather than single N-glycan structures obtained in this clinical glycomics study can serve as a basis for further development of a blood-based diagnostic test., (© 2020 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Body fluid N-glycome analysis as well as glyco-proteoform profiling of existing protein biomarkers potentially provides a stratification layer additional to quantitative, diagnostic protein levels. For clinical omics applications, the collection of a dried blood spot (DBS) is increasingly pursued as an alternative to sampling milliliters of peripheral blood. Here we evaluate DBS cards as a blood collection strategy for protein N-glycosylation analysis aiming for high-throughput clinical applications. A protocol for facile N-glycosylation profiling from DBS is developed that includes sialic acid linkage differentiation. This protocol is based on a previously established total plasma N-glycome mass spectrometry (MS) method, with adjustments for the analysis of DBS specimens. After DBS-punching and protein solubilization N-glycans are released, followed by chemical derivatization of sialic acids and MS-measurement of N-glycan profiles. With this method, more than 80 different glycan structures are identified from a DBS, with RSDs below 10% for the ten most abundant glycans. N-glycan profiles of finger-tip blood and venous blood are compared and short-term stability of DBS is demonstrated. This method for fast N-glycosylation profiling of DBS provides a minimally invasive alternative to conventional serum and plasma protein N-glycosylation workflows. With simplified blood sampling this DBS approach has vast potential for clinical glycomics applications., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

High-throughput mass spectrometry (MS) glycomics is an emerging field driven by technological advancements including sample preparation and data processing. Previously, we reported an automated protocol for the analysis of N-glycans released from plasma proteins that included sialic acid derivatization with linkage-specificity, namely, ethylation of α2,6-linked sialic acid residues and lactone formation of α2,3-linked sialic acids. In the current study, each step in this protocol was further optimized. Method improvements included minimizing the extent of side-reaction during derivatization, an adjusted glycan purification strategy and mass analysis of the released N-glycans by ultrahigh resolution matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance MS. The latter resolved peak overlap and simplified spectral alignment due to high mass measurement precision. Moreover, this resulted in more confident glycan assignments and improved signal-to-noise for low-abundant species. The performance of the protocol renders high-throughput applications feasible in the exciting field of clinical glycomics.

Background & Aims: Biomarkers are needed for early detection of Crohn's disease (CD) and ulcerative colitis (UC) or to predict patient outcomes. Glycosylation is a common and complex posttranslational modification of proteins that affects their structure and activity. We compared plasma N-glycosylation profiles between patients with CD or UC and healthy individuals (controls)., Methods: We analyzed the total plasma N-glycomes of 2635 patients with inflammatory bowel diseases and 996 controls by mass spectrometry with a linkage-specific sialic acid derivatization technique. Plasma samples were acquired from 2 hospitals in Italy (discovery cohort, 1989 patients with inflammatory bowel disease [IBD] and 570 controls) and 1 medical center in the United States (validation cohort, 646 cases of IBD and 426 controls). Sixty-three glycoforms met our criteria for relative quantification and were extracted from the raw data with the software MassyTools. Common features shared by the glycan compositions were combined in 78 derived traits, including the number of antennae of complex-type glycans and levels of fucosylation, bisection, galactosylation, and sialylation. Associations of plasma N-glycomes with age, sex, CD, UC, and IBD-related parameters such as disease location, surgery and medication, level of C-reactive protein, and sedimentation rate were tested by linear and logistic regression., Results: Plasma samples from patients with IBD had a higher abundance of large-size glycans compared with controls, a decreased relative abundance of hybrid and high-mannose structures, lower fucosylation, lower galactosylation, and higher sialylation (α2,3- and α2,6-linked). We could discriminate plasma from patients with CD from that of patients with UC based on higher bisection, lower galactosylation, and higher sialylation (α2,3-linked). Glycosylation patterns were associated with disease location and progression, the need for a more potent medication, and surgery. These results were replicated in a large independent cohort., Conclusions: We performed high-throughput analysis to compare total plasma N-glycomes of individuals with vs without IBD and to identify patterns associated with disease features and the need for treatment. These profiles might be used in diagnosis and for predicting patients' responses to treatment., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Symptoms of rheumatoid arthritis (RA) improve during pregnancy, a phenomenon that was found to be associated with N -glycosylation changes of immunoglobulin G. Recent advances in high-throughput glycosylation analysis allow the assessment of the N -glycome of human sera as well. The aim of this study was to identify new protein N -glycosylation properties that associate with changes in RA disease activity during and after pregnancy. A longitudinal cohort of serum samples was collected during 285 pregnancies (32 control individuals and 253 RA patients). Per individual one sample was collected before conception, three during pregnancy, and three after delivery. Released serum protein N -glycans were measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after employing chemical modification of the sialic acids to allow discrimination of sialic acid linkage isomers. Serum protein N -glycosylation showed strongly modified during pregnancy, with similar changes visible in control individuals and RA pregnancies. Namely, a decrease in bisection and an increase in galactosylation in diantennary glycans were found, as well as an increase in tri- and tetraantennary species and α2,3-linked sialylation thereof. The change in RA disease activity [DAS28(3)-CRP] proved negatively associated with the galactosylation of diantennary N -glycans, and positively with the sialylation of triantennary fucosylated species (A3FGS). While the protein source of the novel finding A3FGS is thus far unknown, its further study may improve our understanding of the etiology of RA disease severity.

Glycosylation is a post-translational modification of key importance with heterogeneous structural characteristics. Previously, we have developed a robust, high-throughput MALDI-TOF-MS method for the comprehensive profiling of human plasma N-glycans. In this approach, sialic acid residues are derivatized with linkage-specificity, namely the ethylation of α2,6-linked sialic acid residues with parallel lactone formation of α2,3-linked sialic acids. In the current study, this procedure was used as a starting point for the automation of all steps on a liquid-handling robot system. This resulted in a time-efficient and fully standardized procedure with throughput times of 2.5 h for a first set of 96 samples and approximately 1 h extra for each additional sample plate. The mass analysis of the thus-obtained glycans was highly reproducible in terms of relative quantification, with improved interday repeatability as compared to that of manual processing.

A fully automated workflow was developed and validated for simultaneous quantification of the cardiovascular disease risk markers apolipoproteins A-I (apoA-I) and B-100 (apoB-100) in clinical sera. By coupling of stable-isotope standards and capture by anti-peptide antibodies (SISCAPA) for enrichment of proteotypic peptides from serum digests to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS detection, the standardized platform enabled rapid, liquid chromatography-free quantification at a relatively high throughput of 96 samples in 12h. The average imprecision in normo- and triglyceridemic serum pools was 3.8% for apoA-I and 4.2% for apoB-100 (4 replicates over 5 days). If stored properly, the MALDI target containing enriched apoA-1 and apoB-100 peptides could be re-analyzed without any effect on bias or imprecision for at least 7 days after initial analysis. Validation of the workflow revealed excellent linearity for daily calibration with external, serum-based calibrators (R(2) of 0.984 for apoA-I and 0.976 for apoB-100 as average over five days), and absence of matrix effects or interference from triglycerides, protein content, hemolysates, or bilirubins. Quantification of apoA-I in 93 normo- and hypertriglyceridemic clinical sera showed good agreement with immunoturbidimetric analysis (slope = 1.01, R(2) = 0.95, mean bias = 4.0%). Measurement of apoB-100 in the same clinical sera using both methods, however, revealed several outliers in SISCAPA-MALDI-TOF-MS measurements, possibly as a result of the lower MALDI-TOF-MS signal intensity (slope = 1.09, R(2) = 0.91, mean bias = 2.0%). The combination of analytical performance, rapid cycle time and automation potential validate the SISCAPA-MALDI-TOF-MS platform as a valuable approach for standardized and high-throughput quantification of apoA-I and apoB-100 in large sample cohorts., (Copyright © 2015 Elsevier Inc. All rights reserved.)

For large-scale and standardized applications in mass spectrometry- (MS-) based proteomics automation of each step is essential. Here we present high-throughput sample preparation solutions for balancing the speed of current MS-acquisitions and the time needed for analytical workup of body fluids. The discussed workflows reduce body fluid sample complexity and apply for both bottom-up proteomics experiments and top-down protein characterization approaches. Various sample preparation methods that involve solid-phase extraction (SPE) including affinity enrichment strategies have been automated. Obtained peptide and protein fractions can be mass analyzed by direct infusion into an electrospray ionization (ESI) source or by means of matrix-assisted laser desorption ionization (MALDI) without further need of time-consuming liquid chromatography (LC) separations.

Aim: Colorectal cancer (CRC) screening programmes detect early cancers but unfortunately have limited sensitivity and specificity. Mass spectrometry-based determination of serum peptide and protein profiles provides a new approach for improved screening., Method: Serum samples were obtained from 126 CRC patients before treatment and 277 control individuals. An additional group of samples from 50 CRC patients and 82 controls was used for validation. Peptide and protein enrichments were carried out using reverse-phase C18 and weak-cation exchange magnetic beads in an automated solid-phase extraction and spotting procedure. Profiles were acquired on a matrix-assisted laser desorption/ionization time-of-flight system. Discriminant rules using logistic regression were calibrated for the peptide and protein signatures separately, followed by combining the classifications to obtain double cross-validated predicted class probabilities. Results were validated on an identical patient set., Results: A discriminative power was found for patients with CRC representative for all histopathological stages compared with controls with an area under the curve of 0.95 in the test set (0.93 for the validation set) and with a high specificity (94-95%)., Conclusion: The study has shown that a serum peptide and protein biomarker signature can be used to distinguish CRC patients from healthy controls with high discriminative power. This relatively simple and cheap test is promising for CRC screening., (Colorectal Disease © 2014 The Association of Coloproctology of Great Britain and Ireland.)

In order to balance the speed of analytical sample preparation procedures with mass spectrometry (MS)-based clinical proteomics the application of high-throughput robotic systems for body fluid workup is essential. In this paper we describe the implementation of various solid-phase extraction (SPE) sample preparation protocols on two different platforms, namely: 1) Magnetic bead-based SPE of peptides and proteins from body fluids on a Hamilton liquid handling workstation; 2) Cartridge-based SPE on a SPARK Symbiosis system. All SPE protocols were optimized for MS-based proteomics and compared with respect to obtained peptide- and protein profiles. Throughput numbers that were achieved in a 24 hour time frame for the sample workup procedures were more than 700 samples for the magnetic bead-based method and over 1000 samples for the cartridge-based method., (Copyright © 2012. Published by Elsevier B.V.)

The continuous efforts to find new prognostic or diagnostic biomarkers have stimulated the use of mass spectrometry (MS) profiles in a clinical setting. In the early days (about one decade ago), a single low-resolution mass spectrum derived from an individual's body fluid was used for comparative studies. However, a peptide profile of a complex mixture is most informative when recorded on an ultrahigh resolution instrument such as a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. In this study we show the benefits of the ultrahigh resolving power and the high mass accuracy and precision provided by an FTICR mass spectrometer equipped with a 15-tesla magnet. The ultrahigh-resolution data not only allow assignment of fragment ions with high charge states (4+, 5+) but also enhance confidence of human serum peptide identifications from tandem MS experiments. This is exemplified with collision-induced dissociation (CID) and electron transfer dissociation (ETD) data of middle-down-sized endogenous or protein-breakdown peptides that are of interest in biomarker discovery studies.

Schistosomiasis is a parasitic infection that is endemic in many developing countries in the tropics and subtropics afflicting more than 207 million people primarily in rural areas. After malaria, it is the second most important parasitic infection in terms of socio-economic and public health. Investigation of the host-parasite interaction at the molecular level and identification of biomarkers of infection and infection-related morbidity would be of value for improved strategies for treatment and morbidity control. To this end, we conducted a nuclear magnetic resonance (NMR) based metabonomics study involving a well-characterized cohort of 447 individuals from a rural area in Uganda near Lake Victoria with a high prevalence of Schistosoma mansoni, a species predominantly occurring in Africa including Madagascar and parts of South America. Cohort samples were collected from individuals at five time-points, before and after (one or two times) chemotherapy with praziquantel (PZQ). Using supervised multivariate statistical analysis of the recorded one-dimensional (1D) NMR spectra, we were able to discriminate infected from uninfected individuals in two age groups (children and adults) based on differences in their urinary profiles. The potential molecular markers of S. mansoni infection were found to be primarily linked to changes in gut microflora, energy metabolism and liver function. These findings are in agreement with data from earlier studies on S. mansoni infection in experimental animals and thus provide corroborating evidence for the existence of metabolic response specific for this infection.

The term molecular histology has been used to convey the potential of imaging mass spectrometry to describe tissue by its constituent peptides and proteins, and to link this with established histological features. The low throughput of imaging mass spectrometry has been one of the factors inhibiting a full investigation of the clinical potential of molecular histology. Here we report the development of an automated set-up, consisting of a controlled environment sample storage chamber, a sample loading robot, and a MALDI-TOF/TOF mass spectrometer, all controlled by a single user interface. The automated set-up is demonstrated to have the positional stability and experimental reproducibility necessary for its clinical application., (Copyright 2009 Elsevier B.V. All rights reserved.)

We introduce mass spectrometry proteomic research for diagnosis from a clinical perspective, with special reference to early-stage breast cancer detection. The nature of SELDI and MALDI mass spectrometric measurement is discussed. We explain how the mass spectral data arising from this technology may be viewed as a new data type. Some of the properties of the data are discussed and we show how such spectra may be interpreted. Sample preprocessing for mass spectrometry is introduced and a literature review of research in clinical proteomics is presented. Finally, we provide a detailed description of the study design on the breast cancer case-control study which is investigated in this special issue.

Serum protein profiling is a promising approach for classification of cancer versus non-cancer samples. The objective of our study was to assess the feasibility of mass spectrometry based protein profiling for the discrimination of colorectal cancer (CRC) patients from healthy individuals. In a randomized block design, pre-operative serum samples obtained from 66 colorectal cancer patients and 50 controls were used to generate MALDI-TOF protein profiles. After pre-processing of the spectra, linear discriminant analysis with double cross-validation was used to classify protein profiles. A total recognition rate (92.6%), sensitivity (95.2%) and specificity (90.0%) for the detection of CRC were shown. The area under the curve of the classifier was 97.3%, and demonstrated the high, significant separation power of the classifier. Double cross-validation shows that classification can be attributed to information in the protein profile. Although preliminary, the high sensitivity and specificity indicate the potential usefulness of serum protein profiles for the detection of colorectal cancer.

Protein profiling with mass spectrometry is a promising approach for classification and identification of biomarkers; however, there is debate about measurement quality and reliability. Here, we present a pipeline for preprocessing, statistical data analysis and presentation. Serum samples of 16 healthy individuals are used to generate protein profiles with high-resolution MALDI-TOF after isolation of peptides with C8 magnetic beads. Analysis of variance was performed after binning, baseline correction and normalization of the mean spectra. Relative variations in the spectra are expressed as coefficient of variation, which depending on the respective preanalytical variation parameter investigated, was found to range between 0.15 and 0.67 in this study. With this novel method, the reproducibility of our protein profiling procedure could be quantified. We showed that circadian rhythm and the number of freeze-thaw cycles had relatively limited influence on serum protein profiles, whereas the period between collection and serum centrifugation had a more pronounced effect.

Rhizobium leguminosarum strain RBL5523 is able to form nodules on pea, but these nodules are ineffective for nitrogen fixation. The impairment in nitrogen fixation appears to be caused by a defective infection of the host plant and is host specific for pea. A Tn5 mutant of this strain, RBL5787, is able to form effective nodules on pea. We have sequenced a 33-kb region around the phage-transductable Tn5 insertion. The Tn5 insertion was localized to the 10th gene of a putative operon of 14 genes that was called the imp (impaired in nitrogen fixation) locus. Several highly similar gene clusters of unknown function are present in Pseudomonas aeruginosa, Vibrio cholerae, Edwardsiella ictaluri, and several other animal pathogens. Homology studies indicate that several genes of the imp locus are involved in protein phosphorylation, either as a kinase or dephosphorylase, or contain a phosphoprotein-binding module called a forkhead-associated domain. Other proteins show similarity to proteins involved in type III protein secretion. Two dimensional gel electrophoretic analysis of the secreted proteins in the supernatant fluid of cultures of RBL5523 and RBL5787 showed the absence in the mutant strain of at least four proteins with molecular masses of approximately 27 kDa and pIs between 5.5 and 6.5. The production of these proteins in the wild-type strain is temperature dependent. Sequencing of two of these proteins revealed that their first 20 amino acids are identical. This sequence showed homology to that of secreted ribose binding proteins (RbsB) from Bacilus subtilis and V. cholerae. Based on this protein sequence, the corresponding gene encoding a close homologue of RbsB was cloned that contains a N-terminal signal sequence that is recognized by type I secretion systems. Inoculation of RBL5787 on pea plants in the presence of supernatant of RBL5523 caused a reduced ability of RBL5787 to nodulate pea and fix nitrogen. Boiling of this supernatant before inoculation restored the formation of effective nodules to the original values, indicating that secreted proteins are indeed responsible for the impaired phenotype. These data suggest that the imp locus is involved in the secretion to the environment of proteins, including periplasmic RbsB protein, that cause blocking of infection specifically in pea plants.

Background: In response to reported wound complication rates of 19% to 43% for traditional saphenous vein harvest, several minimally invasive vein harvest (MIVH) techniques have been developed. The purpose of this investigation is to determine the effectiveness of one such MIVH technology, the Genzyme SaphLITE Retractor System (Genzyme Biosurgery, Cambridge, MA)., Methods: Since May 2000, saphenectomy was undertaken in 305 coronary artery bypass graft (CABG) patients using SaphLITE in a prospective, nonrandomized trial at three centers. Patients were assessed for wound healing (ASEPSIS tool) and incisional pain (numeric scale) through the postoperative visit. Harvest times, incision lengths, and vein lengths were recorded., Results: ASEPSIS indicated satisfactory healing in 96.0%. Infection rate was 1.3% with four patients requiring antibiotics and debridement of one incision. Of hospitalized patients, 85.4% had no or minimal affected leg pain. Additional mean data include: harvest time 43.4 +/- 17.6 minutes, incision number 3.0 +/- 1.2, incision length 2.9 +/- 1.4 cm, and vein length 46.0 +/- 15.2 cm., Conclusions: SaphLITE provides an effective alternative to traditional saphenous vein harvest, with improved wound healing, decreased pain, and acceptable harvest times.

The symbiosis between Rhizobium bacteria and their host plants is dependent on the specific recognition of signal molecules produced by each partner. Many players in the signal exchange have been identified. Among them are signal molecules such as flavonoids, LCOs, auxin, cytokinin, ethylene and uridine and genes such as Enod40, Enod2 and Enod12. Their interconnection, however, is only starting to be understood. The most recent insights into their interconnection include: advances in the use of transgenic leguminous plants containing reporter gene constructs for studying the effect of the signal molecules; novel methods for delivery of signal molecules using ballistic microtargeting; and the discovery of the role of chitin oligosaccharides in animal embryogenesis.

Erythroleukemia K562 cells and lentil (Lens culinaris) root protoplasts have been subjected to pore-forming electric fields suitable for transfection experiments. Evidence is presented that the amount of hydroperoxides formed in cell membranes of both cell-types is a function of field strength applied. On the other hand, the electroporation-induced lipid peroxidation paralleled the enhancement of membrane permeability and was associated with greater membrane fluidity. The membrane hydroperoxides formed upon electric shock enhanced cell luminescence, and lipoxygenase activity appeared to be involved in the process. Electroporation of prokaryotic cells of Escherichia coli also enhanced light emission, which was higher in lipoxygenase-expressing clones.

Eighteen dogs underwent transmural left ventricular biopsies for adenosine triphosphate and suturing of the noncoronary cusp, creating valvular aortic stenosis. Three months after aortic stenosis and the subsequent development of left ventricular hypertrophy, animals underwent repeat transmural left ventricular biopsies followed by total myocardial ischemia at 37 degrees C. Left ventricular tissue samples for adenosine triphosphate and lactate levels were determined at 15-minute intervals and compared with 15 control animals. No significant difference between subendocardial and subepicardial adenosine triphosphate levels was found between left ventricular samples taken before left ventricular hypertrophy and 3 months after left ventricular hypertrophy. Significant differences in adenosine triphosphate utilization occurred between subendocardial and subepicardial layers in control and left ventricular hypertrophy myocardium, however. The gradient between the subendocardium and the subepicardium was significantly increased by left ventricular hypertrophy (p less than 0.05). Significant differences also occurred within the same layer when left ventricular hypertrophy and control groups were compared. During total ischemia, lactate concentration was significantly greater within the subendocardium than within the subepicardium in left ventricular hypertrophy. The onset of ischemic contracture was 48.2 +/- 2.1 minutes in left ventricular hypertrophy versus 62.3 +/- 1.8 minutes in control hearts (p less than 0.01). Subendocardial intramyocardial pressure increased significantly earlier than subepicardial in both left ventricular hypertrophy and control hearts. Adenosine triphosphate was used, and lactate accumulated more rapidly in animals with a more pronounced hemodynamic gradient. These data show that after left ventricular hypertrophy, adenosine triphosphate stores in the subendocardium and the subepicardium are unchanged from control values, yet the rates of adenosine triphosphate utilization and lactate accumulation during total ischemia are significantly increased. Furthermore, the subendocardial to subepicardial gradient of adenosine triphosphate utilization during ischemia found in normal hearts is markedly increased by left ventricular hypertrophy.

Prehospital cardiopulmonary resuscitation combined with endotracheal intubation, vigorous fluid resuscitation, and rapid transport can be effective in resuscitating trauma patients in cardiopulmonary arrest. Survival does not correlate with the injury severity score or transport time once the patient has arrested but does correlate with the mechanism of injury, endotracheal intubation, and placement of intravenous lines.

Over the past 47 years (1937 to 1984), a total of 127 patients with esophageal perforation or rupture were evaluated at Duke Medical Center or the Durham Veterans Administration Medical Center. In 13 patients, the diagnosis was established at the time of autopsy and in the remaining 114, the diagnosis was established clinically. The etiology, radiological findings, underlying esophageal disease, time interval between onset of symptoms and therapy, and eventual outcome were evaluated. Patients with anastomotic leaks and those in whom carcinoma resulted in perforation or fistula were excluded. Iatrogenic causes were responsible for 55% of perforations, followed by spontaneous rupture in 15%, foreign body perforation in 14%, and traumatic perforation in 10%. Of the 127 patients, 114 underwent treatment involving primary closure (43%), drainage alone (28%), resection (9%), or nonoperative therapy (20%). The overall mortality among these 114 patients was 21%. Fourteen patients sustained a major complication requiring additional operative intervention. The overall mortality among patients requiring reoperation was 57%. Survival was significantly influenced by a delay in treatment of greater than 24 hours. With the exception of nonoperative therapy, survival was improved for all forms of treatment instituted within 24 hours. Primary closure within 24 hours resulted in the most favorable outcome (92% survival). In addition to early treatment, other factors associated with a favorable outcome included traumatic perforation (100% survival), foreign-body perforations (94% survival), and iatrogenic causes (80% survival). Spontaneous rupture resulted in the lowest survival (37%). The incidence of esophageal perforation has increased dramatically since 1967.(ABSTRACT TRUNCATED AT 250 WORDS)